Abstract B024: Targeting tmCLIC1 to overcome resistance across heterogeneous glioblastoma subtypes by exploiting metabolic vulnerabilities with synergistic combinatorial therapy

The overall goal of this work was to assess the ability of Natural Killer cells to kill cultures of patient-derived glioblastoma cells. Herein we report impressive levels of NK-92 mediated killing of various patient-derived glioblastoma cultures observed at ET (effector: target) ratios of 5:1 and 1:1. This enabled direct comparison of the degree of glioblastoma cell loss across a broader range of glioblastoma cultures. Importantly, even at high ET ratios of 5:1, there are always subpopulations of glioblastoma cells that prove very challenging to kill that evade the NK-92 cells. Of value in this study has been the application of ECIS (Electric Cell-Substrate Impedance Sensing) biosensor technology to monitor the glioblastoma cells in real-time, enabling temporal assessment of the NK-92 cells. ECIS has been powerful in revealing that at higher ET ratios, the glioblastoma cells are acutely sensitive to the NK-92 cells, and the observed glioblastoma cell death is supported by the high-content imaging data. Moreover, long-term ECIS experiments reveal that the surviving glioblastoma cells were then able to grow and reseed the culture, which was evident 300-500 h after the addition of the NK-92 cells. This was observed for multiple glioblastoma lines. In addition, our imaging provides evidence that some NK-92 cells appear to be compromised early, which would be consistent with potent evasive mechanisms by the glioblastoma tumour cells. This research strongly highlights the potential for NK-92 cells to kill glioblastoma tumour cells and provides a basis to identify the mechanism utilised by the surviving glioblastoma cells that we now need to target to achieve maximal cytolysis of the resistant glioblastoma cells. It is survival of the highly resistant glioblastoma clones that results in tumour relapse.

Recent genomic studies on the glioblastoma (GBM) subtypes (e.g., mesenchymal, proneural, and classical) pave a way for effective clinical treatments of the recurrent brain tumor. However, identification of the GBM subtype is complicated by the intratumoral heterogeneity that results in coexistence of multiple subtypes within the tissue specimen. Here, we present the use of hyperspectral stimulated Raman scattering (SRS) microscopy for rapid, label-free molecular assessment of GBM intratumoral heterogeneity with submicron resolution. We develop a unique label-free Raman imaging diagnostic platform consisting of the spectral focusing hyperspectral SRS imaging of the large-area GBM tissue specimens, SRS images, and spectrum retrieval using the multivariate curve resolution algorithm and subtype classification based on the quadratic support vector machine model for rapid molecular subtyping of GBMs. Both the stain-free SRS histological images and 2D subtype maps can be obtained within 20-30 min which is superior to the days of the conventional single-cell RNA sequencing. While the SRS histology assesses the demyelination status as a new diagnostic feature, the SRS mapping provides a new insight into intratumoral heterogeneity across GBM tissue specimens. We find that the major proportions of the GBM tissues agree with the diagnostic results of the genomic analysis, but nontrivial portions of the remaining SRS image tiles in the specimens are found to belong to other molecular subtypes, implying the substantial degree of GBM heterogeneity. The rapid SRS imaging diagnostic platform developed has shown the ability of unveiling tumor heterogeneity in GBM tissues accurately, which would promote the improvement of the GBM-targeted therapy in near future.

Purpose: Glioblastoma (GBM) exhibits functional heterogeneity and therapeutic resistance across disease stages. We developed a 3D magnetic spheroid platform to assess invasion, stem cell plasticity, and therapeutic response in newly diagnosed and recurrent patient-derived gliomaspheres under physiologically relevant hypoxic conditions. Methods: Gliomasphere migration was monitored by live-cell imaging under hypoxia. Stem cell plasticity was assessed by CD44 expression using laser scanning confocal microscopy, while basal MCL-1 levels and CDK inhibitor targets were analysed by Western blotting. Cell viability and apoptosis were evaluated using WST-1 and Caspase-3/7 Glo assays. Invasion dynamics in response to Temozolomide (TMZ) and novel CKIs (CYC065 & THZ1) were assessed across disease stages. Results: Newly diagnosed gliomaspheres exhibited dynamic CD44 plasticity, whereas recurrent gliomaspheres maintained stable CD44 expression. In unmatched samples, newly diagnosed tumors were more invasive than recurrent tumors; this difference was not observed in patient-matched pairs. Across all stages and oxygen conditions, CDK inhibitors far more effectively suppressed invasion and induced apoptosis than TMZ, independent of CD44 status. CKI activity was associated with MCL-1 downregulation and inhibition of CDK2/7/9, and was maintained despite fluctuations in BCL-2 family proteins under hypoxia, confirmed by reduced viability and increased Caspase-3/7 activity. Conclusions: This 3D magnetic spheroid platform captures spatially resolved functional heterogeneity in glioblastoma and enables precise evaluation of invasion and therapeutic response. Novel CDK inhibitors robustly suppress invasion and induce apoptosis across disease stages and oxygen conditions, highlighting their potential as broadly effective therapeutics. This work demonstrates how spatial 3D modeling can uncover hypoxia driven plasticity and inform translational strategies in glioblastoma. Generative artificial intelligence was used to assist with language editing and clarity of the abstract; all scientific content and conclusions were determined by the authors. Citation Format: Niamh M. Scully, Brenton L. Cavanagh, Heiko Duessmann, Jochen H.M. Prehn, Brona M. Murphy. 3D magnetic gliomasphere modeling reveals CDK inhibitor mediated suppression of migration in newly diagnosed and recurrent glioblastoma under hypoxic conditions [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr B029.

Glioblastoma (GBM) is the most common and lethal primary brain malignancy in adults, with a median survival of 12–14 months and a 5-year survival rate of ∼6%. Despite decades of effort, patient outcomes have improved minimally, underscoring an urgent need for more effective and targeted therapies. We identified the bromodomain-containing chromatin regulator BRD8 as an essential dependency in TP53-wild-type (TP53WT) GBM, which comprises ∼71% of cases. BRD8 sustains malignancy by disabling p53-mediated tumor suppression through a previously unrecognized epigenetic mechanism: BRD8 reprograms the p53 regulatory network via the EP400 histone acetyltransferase complex and bromodomain-directed occupancy of the histone variant H2AZ at p53 target genes, enforcing a repressive chromatin state that blocks p53-dependent transactivation. Targeting BRD8 remodels chromatin by evicting H2AZ and increasing accessibility, and restores p53 binding and transcriptional activity. This reactivation of p53 restores gene expression, induces cell-cycle arrest, suppresses gliomagenesis, and prolongs survival in TP53WT GBM xenograft models. Our recent work further demonstrates that pharmacological targeting of BRD8 selectively restores p53 tumor-suppressive activity in TP53WT GBM cells, but not in non-malignant brain cells. Notably, BRD8 inhibition synergizes with multiple therapeutic pathways and enhances the efficacy of standard-of-care GBM treatments in patient-derived TP53WT GBM models. Together, these findings uncover a previously unappreciated epigenetic mechanism by which GBM cells evade p53-mediated tumor suppression, establish BRD8 as a tractable epigenetic vulnerability in TP53WT GBM, and nominate rational combination therapeutic strategies for GBM treatment. This work reveals new principles of chromatin-based tumor suppression and offers promising therapeutic avenues for this devastating disease. Citation Format: Xueqin Sherine Sun. An epigenetic dependency on BRD8 in TP53-wild-type glioblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr B008.

Objective: IDH-wildtype glioblastoma (GBM) is the most aggressive brain tumor in adults. Notable for substantial inter- and intra-tumoral heterogeneity, GBM is highly resistant to standard-of-care treatment. Emerging spatial omics technologies, such as GeoMx Digital Spatial Profiling (DSP), offer novel approaches to interrogate the molecular features of regionally defined cellular ecosystems in cancer. Here, we leverage DSP to study whole-transcriptome gene expression signatures in a spatially defined manner across five IDH-wildtype GBMs. Methods: Formalin-fixed paraffin-embedded tumors were assessed histologically and three spatially distinct areas from each tumor were used to construct a custom “spatial” tissue microarray. From each set of three areas, we identified nine spatially distinct regions of interest (ROIs) for bulk transcriptomic analysis according to two histopathological features: distribution (cellular tumor core or tumor periphery) and vascular proximity (near or far). We evaluated normalized target expression data statistically using linear mixed modeling (LMM) with Benjamini-Hochberg correction. Results: 44 profiled ROIs and 8531 transcriptomic targets were analyzed following quality control trimming. 3D principal component analysis and unsupervised hierarchical clustering analysis showed clustering according to tumor specimen as well as distribution (core versus periphery), thereby substantiating the presence of inter- and intra-tumoral transcriptional heterogeneity. LMM-based differential gene expression analysis identified distinct molecular signatures when comparing core and periphery. Specifically, we observed elevated log2-transformed expression of migration and proliferation markers including LAMC1 (p=0.00297) and FABP7 (p=0.00434) in core ROIs and neurodevelopmental markers including NTRK2 (p=0.00734) and DAAM2 (p=0.00263) in periphery ROIs. Additionally, reactome analysis demonstrated considerable enrichment for the aberrant programmed cell death pathway (R-HSA-9645723, coverage=71.84%) in core ROIs and the MHC class II antigen presentation pathway (R-HSA-2132295, coverage=69.11%) in periphery ROIs. Interestingly, differential gene expression analysis did not reveal any biologically significant targets when subsetting for vascular proximity within cellular tumor core ROIs. However, reactome analysis of this regional subset demonstrated upregulation of various cellular proliferation pathways (G2/M transition, APC/C activity, DNA synthesis, etc.) within vessel-near ROIs when compared with vessel-far ROIs. Conclusions: We preliminarily characterize the molecular heterogeneity of GBM using a spatially guided approach. Ongoing analyses are focused on dataset validation, mixed cell deconvolution, further regional genotyping, and potential biomarkers. Citation Format: Adam Luo, Kelly Casella, Shivang Sharma, Ezra Baraban, Calixto-Hope Lucas, Eugene Shenderov. Utilizing whole-transcriptome digital spatial profiling for glioblastoma clinical biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3766.

<div>Abstract<p>A consequence of the intratumor heterogeneity (ITH) of glioblastoma (GBM) is the susceptibility to treatment-driven evolution. To determine the potential of radiotherapy to influence GBM evolution, we used orthotopic xenografts initiated from CD133<sup>+</sup> GBM stem–like cells (GSC). Toward this end, orthotopic xenografts grown in nude mice were exposed to a fractionated radiation protocol, which resulted in a significant increase in animal survival. Brain tumors from control and irradiated mice were then collected at morbidity and compared in terms of growth pattern, clonal diversity, and genomic architecture. In mice that received fractionated radiation, tumors were less invasive, with more clearly demarcated borders and tumor core hypercellularity as compared with controls, suggesting a fundamental change in tumor biology. Viral integration site analysis indicated a reduction in clonal diversity in the irradiated tumors, implying a decrease in ITH. Changes in clonal diversity were not detected after irradiation of GSCs <i>in vitro</i>, suggesting that the radiation-induced reduction in ITH was dependent on the brain microenvironment. Whole-exome sequencing revealed differences in mutation patterns between control and irradiated tumors, which included modifications in the presence and clonality of driver mutations associated with GBM. Moreover, changes in the distribution of mutations as a function of subpopulation size between control and irradiated tumors were consistent with subclone expansion and contraction, that is, subpopulation evolution. Taken together, these results indicate that radiation drives the evolution of the GSC-initiated orthotopic xenografts and suggest that radiation-driven evolution may have therapeutic implications for recurrent GBM.</p>Significance:<p>Radiation drives the evolution of glioblastoma orthotopic xenografts; when translated to the clinic, this may have therapeutic implications for recurrent tumors.</p></div>

Cells within tumours have diverse genomes, epigenomes, and interact differentially with their surrounding microenvironment generating intratumour heterogeneity, which has critical implications for treating cancer patients. I will present our current approach to understand the intratumour heterogeneity in glioblastoma through the analysis of patient-derived models. I will present examples of how we perform functional analysis to understand genomic alterations in the context of inter- and intra-tumoral heterogeneity taking special interest on their therapeutic implications. Linked to the concept of intratumor heterogeneity in the cellular state of differentiation, I will explain our studies related to cancer-initiating cells in glioblastoma, and I will focus on the TGF-beta pathway which has a critical role in glioblastoma. Citation Format: Joan Seoane. Intratumoral heterogeneity in glioblastoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr IA17. doi:10.1158/1538-7445.CHTME14-IA17

INTRODUCTION: Intratumour heterogeneity (ITH) may account for therapy failure and tumour recurrence in adult glioblastoma multiforme (GBM). Low availability of fresh frozen tissue has hindered characterisation of ITH in paediatric GBM. Here, we propose the application of desorption electrospray ionisation mass spectrometry (DESI-MS) to assess spatial lipidomic profiles of formalin-fixed paraffin-embedded (FFPE) tissue of paediatric GBM. METHOD: Optimisation of DESI-MS has been conducted on FFPE tissue of normal adult brain prior to transfer of the technology to paediatric GBM tissue. Immunohistochemical analyses for proteins associated with lipid biosynthesis delineated distinct intratumour regions. Spatial characterisation of lipids synthesised by these metabolic proteins will permit optimisation of DESI-MS, prior to unbiased lipidomic analyses. RESULTS: Paediatric GBM cell lines grown under lipoprotein-deficient conditions demonstrated greater resilience compared to an adult cell line, indicating distinct lipid metabolism that will be revealed through lipidomic analyses. Proteins associated with lipid biosynthesis demonstrated evidence of ITH in paediatric GBM, with 69% of patients displaying heterogeneous expression of fatty acid synthase that correlated with areas of high cellularity. CONCLUSION: DESI-MS has the potential to characterise early indications of paediatric GBM ITH through characterisation of lipid markers associated with distinct intratumour regions. Proteins associated with the synthesis of identified lipid markers may represent potential therapeutic targets. We anticipate the establishment of an optimised DESI-MS method that will facilitate lipidomic studies of other paediatric brain tumours.

Resistance to therapy is a major limitation towards the cure of irresectable cancer. Cellular senescence, induced by chemotherapy and radiotherapy, can drive therapy resistance in vivo. Senescence is a tumor suppressive mechanism, but senescent cells can secrete a range of proteins that ironically promote tumor growth, migration and metastazation and therapy resistance. Recent evidence has shown that genetic removal of senescent cells indeed causes a strong reduction in tumor growth and metastasis formation. Unfortunately however, therapeutic options to remove senescent cells are currently lacking. Here, we show the development and optimization of a biochemical compound, TASC1, that potently and selectively kills senescent cells in vitro and in vivo and lowers organ toxicity in a mouse model employed to address the off-target effects of cancer therapy. This encouraged us to test the effect of TASC1 against cancer therapy resistance. We focused on Glioblastoma multiforme (GBM), the most aggressive and lethal stage of glioma (III-IV) with a median survival of less than 15 months. Treatment of GBM involves a combination of surgery, radiotherapy and adjuvant chemotherapy (Temozolomide). Response to radiotherapy is generally strong, but resistance occurs rapidly, after which currently no successful follow-up treatment exists. We observed radiation therapy to induce senescence in astrocytes and senescence-like effects in GBM cells, without affecting their progression. Strikingly, while both TASC1 and radiation at various doses independently failed to reduce growth of these GBM cells, the combination of TASC1 with radiation showed a potent synthetic lethal response. Similar effects were seen for other types of cancer and their respective therapies. Thus, TASC1 is a potent adjuvant to (re)sensitize therapy-resistance metastatic cancer cells to treatment. Citation Format: Marjolein P. Baar, Diana A. Putavet, Joris Pothof, Jan H.J. Hoeijmakers, Peter L.J. de Keizer. TASC1, a selective anti-senescence therapeutic which potently and selectively counteract resistance to chemo- and radiotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2843.

Background: Glioblastoma (GBM) is a universally fatal brain tumor. Tumor-associated macrophages (TAMs) comprise up to 40% of GBM tumors and predominantly demonstrate an M2 phenotype, favoring tumor growth while suppressing host immune response. Previously, we showed that in p50-/- mice lacking the repressive NF-κB subunit, GBM TAMs are biased toward the pro-inflammatory M1 phenotype, limiting GBM tumor growth by increasing activated tumor T cells. The present study aims to determine whether adoptively transferred immature myeloid cells lacking p50 (p50-IMC) localize to mouse GBM tumors and slow tumor growth. Methods: Lineage-negative bone marrow (BM) cells were isolated from C57BL/6 (B6) p50-/- mice, expanded, and treated with Macrophage-Colony Stimulating Factor (M-CSF) for 24h to produce p50-IMC. BM-derived myeloid cells from both wild-type (WT) and p50-/- mice were subjected to analysis of M1 (TNFα, IL-12b) and M2 (Fizz1, Mannose Receptor) markers via quantitative PCR after exposure to M-CSF for 24h or after M2-polarization via five days in M-CSF and then two days in IL-4. B6-derived GL261-Luc GBM cells were inoculated intracranially into wild-type B6 mice, and four days post-implantation, mice were given 5-fluorouracil to deplete endogenous leukocytes. Mice then received either no additional treatment or three doses of ten million p50-IMCs 9-, 11-, and 14-days post-implantation, and tumor growth was monitored via bioluminescence, measured following luciferin injection using the Spectrum in vivo imaging system. The number of p50-IMCs localizing to the GBM-containing brain was determined by CFSE-dye-labeling of IMC followed by flow cytometry analysis 24h after injection. Results: Both TNFα and IL-12b were increased in p50-/- cells after 5 days M-CSF, and increased IL-12b expression was maintained even after M2-polarization with IL-4. Of the 10 million injected p50-IMCs, 0.07% of those cells were found in the brain. Despite such a small number of p50-IMCs localizing to the tissue of interest, inhibition of GBM growth was observed. Mice receiving the p50-IMC treatments (n = 4) demonstrated a 2.3- and 2.4-fold reduction in tumor growth at 15 (p = 0.13) and 18 (p = 0.14) days post-implantation, respectively, compared to control mice (n = 5). At 21 days post-implantation, tumor size was effectively equivalent between the two groups (p=0.76). p50-IMC treatment did not correlate with improved survival. Conclusion: Loss of p50 skews myeloid cells toward a proinflammatory phenotype, even under M2-polarizing conditions. Adoptive transfer of p50-IMC transiently impairs GBM growth in an orthotopic mouse model. Although statistical significance was not attained, this may be due to the small number of animals used in this experiment. Further investigation is needed to evaluate the full potential of p50-IMC as an immunotherapy option for GBM patients. Citation Format: Patrick J. Beck, Theresa Barberi, Alan D. Friedman. Adoptive transfer of NF-kB p50 knockout immature myeloid cells shows a trend towards slower glioblastoma tumor growth in an orthotopic mouse model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4063.

Letrozole (LTZ), a third-generation aromatase inhibitor, appears to be a promising novel therapeutic for the treatment of glioblastoma (GBM). It synergistically enhances the efficacy of the DNA alkylating agent temozolomide, one of the few approved chemotherapeutics for GBM, against patient-derived GBM cells. RNA-seq analysis of tumor from recurrent GBM patients treated with LTZ prior to surgery in a “window of opportunity” phase 0/1 dose escalation clinical trial revealed that LTZ downregulates genes encoding for DNA-damage repair proteins (BRCA2) in a dose dependent manner. Our central hypothesis for this project is that combination of LTZ with targeted therapeutic agents that promote DNA damage results in synergistic effects against GBM. The objective of this study is to evaluate the effects of poly-ADP ribose polymerase (PARP) inhibitors in combination with LTZ against patient-derived GBM cells. Our secondary aim for this project is to delineate the blood-brain barrier (BBB) permeability of PARP inhibitors. Employing patient-derived G43, G75 and JHH-136 GBM lines we assessed the influence of PARP inhibitors (olaparib, pamiparib, veliparib and niraparib) in combination with LTZ on cell viability and neurosphere growth. We then utilized male CD-IGS rats (n=6; body wt., 250g) to assess plasma and brain extracellular fluid (ECF) pharmacokinetics (PK) of PARP inhibitors using cerebral microdialysis. The IC50 values for PARP inhibitors in these assays ranged from 0.06 to 11.31 µM. Treatment in combination with LTZ (0.05 µM), a non-cytotoxic concentration of LTZ in these lines, significantly decreased the IC50 values for PARP inhibitors (0.012 to 0.51 µM). Combination Index analysis indicated that this combination was strongly synergistic. Furthermore, the plasma and brain ECF PK analyses suggested that PARP inhibitors such as pamiparib and niraparib exhibit facile transport across the BBB barrier as reflected in Kp,uu,brain (ratio of AUCecf/AUCplasma, unbound) values of 0.98 and 0.45, respectively. Overall, these results provide a strong foundation for pursuing further development of combination therapy of GBM with LTZ and PARP inhibitors. A careful assessment of the clinical efficacy and safety profile of these agents will further guide shortlisting combination of LTZ for pre-clinical and clinical development. Citation Format: Aniruddha Sunil Karve, Bhavesh Babulal Gabani, Sidharth Nitin Gadgil, Shravani Parag Kulkarni, Gary A. Gudelsky, Pankaj B. Desai. Translational research to facilitate development of novel therapeutic combinations of letrozole for the treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2119.

Intratumor heterogeneity exists in many cancers, with clear evidence of differential gene expression temporo-spatially in glioblastoma multiforme (GBM). MicroRNA (miRNA) profiling studies have identified risk-stratifying signatures in GBM, but it has not been clarified whether microRNA profiles vary in a similar fashion to gene expression within individual GBM tumors. This study utilized primary GBM specimens resected from patients undergoing surgery in a regional UK Neurosurgery unit. Fresh frozen tissue underwent RNA extraction and miScript Human Brain Cancer miRNA PCR Array was used to assess 84 miRNA's expression levels between distinct spatial regions within primary GBMs. miRNA expression was analyzed with quantitative RT-PCR and gene targets for analysis were selected with miRtarget gene prediction software. Protein levels for the in silico predicted targets based on miRNA expression were validated through immunohistochemistry (IHC). A large degree of intratumor heterogeneity was apparent in the samples. Homology existed between individuals with 8 common miRNAs differentially expressed between central and edge regions of tumors. Protein levels of CDK6 and c-KIT nuclear accumulation were significantly elevated at tumor edges relative to core samples in concordance with the microRNA profiling. Intratumor heterogeneity in GBM produces significant miRNA profiling variation depending on spatial location of the sample within the tumor. Therefore, assessment of GBM miRNA signatures requires selective sampling from multiple distinct tumor regions and consideration of the overall patterns of miRNA expression across the entirety of the tumor.

GPR133 (ADGRD1), an adhesion G protein–coupled receptor (GPCR) whose canonical signaling activates GαS-mediated generation of cytosolic cAMP, has been shown to be necessary for the growth of glioblastoma (GBM), a brain malignancy. The extracellular N terminus of GPR133 is thought to be autoproteolytically cleaved into N-terminal and C- terminal fragments (NTF and CTF, respectively). However, the role of this cleavage in receptor activation remains unclear. Here, we used subcellular fractionation and immunoprecipitation approaches to show that the WT GPR133 receptor is cleaved shortly after protein synthesis and generates significantly more canonical signaling than an uncleavable point mutant GPR133 (H543R) in patient-derived GBM cultures and HEK293T cells. After cleavage, the resulting NTF and CTF remain noncovalently bound to each other until the receptor is trafficked to the plasma membrane, where we demonstrated NTF–CTF dissociation occurs. Using a fusion of the CTF of GPR133 and the N terminus of thrombin-activated human protease-activated receptor 1 as a controllable proxy system to test the effect of intramolecular cleavage and dissociation, we also showed that thrombin-induced cleavage and shedding of the human protease-activated receptor 1 NTF increased intracellular cAMP levels. These results support a model wherein dissociation of the NTF from the CTF at the plasma membrane promotes GPR133 activation and downstream signaling. These findings add depth to our understanding of the molecular life cycle and mechanism of action of GPR133 and provide critical insights that will inform therapeutic targeting of GPR133 in GBM.

Introduction: Glioblastoma Multiforme (GBM) is a lethal and highly aggressive cancer where risk factors, other than radiation exposure, are still largely unknown. It has been reported that US black patients have lower brain cancer rates than whites, and this has been ascribed to differences in socio-economic status. No study has analyzed possible risk factors that could explain the observed differences in rates with race. Methods: All cases of pathologically confirmed GBM diagnosed between 1973 and 2010 were extracted from the Surveillance, Epidemiology and End Results (SEER) data base, and age-adjusted incidence rates x 100,000 persons were calculated according to race (blacks, whites, others), and age groups (0-19, 20-49, 50-85+ years). Survival was calculated using Cox proportional hazard models according to race. The NHANES data base was analyzed to assess time since last visit to a dentist (surveyed in 1999 through 2004), performance and frequency of a routine check-up in previous 3 years (surveyed in 1999 through 2002). Results: There were 25,101 GBM patients in the data set, 360 of which were age 0-19 years, 4,234 age 20-49 years, 20,507 age 50-85+ years. GBM incidence rate was 0.1 x 100,000 persons in the younger age group, with no differences between black and white patients. Among patients age 20-49 years the incidence rate was 1.1 x 100,000 in whites and 0.7 x 100,000 in blacks, in patients age 50-85+ years the incidence rate was 9.5 x 100,000 and 4.4 x 100,000. The ratio of the incidence in white patients/black patients was 1, 1.6 and 2.2 respectively in the three age groups, without differences across geographic areas where the diagnosis was performed. Survival was similar in the two race groups: 3 year survival was 6.5% in whites, 9% in blacks; 5year survival was 3.8% and 4.7% respectively. Cancer treatment modalities did not significantly differ between the two races. From the NHANES data base (26,958 subjects, blacks = 6,719), 65.6 % of whites had a dentist visit within the previous year versus 54.3% of blacks (p=0.001). White subjects were significantly more likely to undergo routine check up once or more a year in comparison to black subjects (81.9% vs. 68.3%; p = 0.001). Conclusions: GBM rates continue to be lower in black than white patients. The difference is largest in the older age group, suggesting a relation to the genetic background and subtype of GBM that occurs in patients older than 50 years and/or to environmental exposure. There is no geographic difference in cancer rates arguing against state specific environmental risk factors. Given the overlapping survival observed in black and white patients, it is unlikely that access to care plays a role in the differences in incidence rates. Racial differences in access to dental care and diagnostic radiation exposure should be explored in ad-hoc epidemiologic studies. Citation Format: Emanuela Taioli, Wenqi Gan, Gregory J. Riggins. Health disparities in brain cancer incidence. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr B17. doi:10.1158/1538-7755.DISP13-B17

The RhoA GTPase is thought to play a crucial role in cancer development. Its activation is tightly controlled by numerous guanine nucleotide-exchange factors (GEFs). We previously showed that Syx, a RhoA-GEF, promotes junctional integrity in endothelial cells and governs cell polarity and directed migration in glioblastoma (GBM) cells. GBM is a grade IV glioma with a median survival of approximately 14 months. Importantly, the gene locus of Syx (Plekhg5) is located within chromosome 1p36, a region deleted in oligodendroglioma (a type II/III glioma) and associated with better prognosis, suggesting a role of Syx in GBM aggressiveness. Here we show that Syx is a potential target for the treatment of GBM. Knockdown of Syx resulted in altered expression of cell cycle regulators, including cyclins A, B, and E, cyclin-dependent kinase inhibitors (CDIs), namely p21 and p27, and mitosis modulators CDC20 and survivin. These defects resulted in G2/M arrest and dysfunction of cell division, leading to improper chromosome segregation and DNA double-strand breaks. However, DNA damage response could not be activated effectively for DNA repair, leading to cellular apoptosis. Consistent with these findings, we used an orthotopic patient-derived xenograft GBM model to show that Syx silencing results in decreased tumor growth accompanied by increased animal survival. Agents that cause DNA damage and/or disruption of DNA repair pathways are often used for cancer therapy. We thus tested the possibility of targeting Syx for GBM treatment, and showed that Syx knockdown in combination with temozolomide, a first-line drug for GBM, synergistically inhibit cell growth. In summary, this is the first study showing the role of Syx in GBM cell growth and that targeting this signaling pathway can be beneficial for GBM therapy. Citation Format: Wan-Hsin Lin, Ryan Feathers, Laura Lewis-Tuffin, Panos Anastasiadis. Targeting the RhoA guanine nucleotide-exchange factor Syx for the treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4365.